An integrated informatics and resources platform for Reverse Genetics in dicots (RevGenUK)

In today's changing climate, we are constantly trying to improve crops to achieve more sustainable agricultural practices. To do this, we need to understand in great detail how plants grow and work. Genetics is a powerful tool for helping us to obtain such an understanding. Using it we are able to analyse the whole genetic make-up (the genome) of plants to discover all the genes required for them to develop and operate correctly. We can use this information to study variants that have a defect in a particular gene (or genes) in which we are interested. This helps us to understand how the gene works when it is operating normally and consequently its role in the plant. Some species of plant have had their whole genome sequenced already, but there are still thousands of their genes whose role in the plant is not understood. To obtain plants that bear a defective gene we can treat seeds with chemicals or radiation that damage the DNA coding for that gene. The offspring from these treated seeds will bear a large number of defects in their genomes. By producing a large population of offspring we can make every gene in the genome bear a defect. The problem then is to find the plant in the population that has a defect in the gene in which you are interested. We have developed methods that can sort out defective genes of interest and find the plants that contain them, so we can find out the role of the gene. This whole process is known as reverse genetics. No one chemical or physical means can induce all the defects we need to study particular genes so we need to use several different methods. In this project we will set up a resource for the plant science research community so that they can discover the function of their particular genes of interest. We will set up populations of legumes and brassicas (treated with chemicals or radiation) that contain plants bearing different forms of defective genes. We have developed special methods based on highly efficient machines (sequencers) that can detect the defects when compared to the normal gene. Scientists can then send us information about the gene of interest, for example, a legume gene, and we can then look for defects in their specific gene in our population of thousands of legume plants. We then send them seeds from the plant that they can grow to study the action of the defective gene in that plant. All the information that we gather about our plants and their thousand upon thousands of genes will be stored in a computer database that we will construct especially for this project, although it will be written in such a way that others can use it as well. It will also be available to use on the worldwide web so that a scientist anywhere in the UK or the World can come and browse to see if the database contains information about their gene of interest. The reason for wanting to do this is to improve the ability of crop plants to grow in different environments, especially adverse ones, and to help the farmer work in a sustainable way using less added fertiliser and fewer herbicides and pesticides.

The genome sequences of Arabidopsis and rice have been completed and those of other species including Lotus japonicus, Medicago truncatula and Brassica rapa are currently underway. To uncover the function of the thousands of genes that will be discovered by these initiatives will be a major challenge. Reverse genetics uses genome sequence to permit the recovery of mutants in target genes for use in functional genomics. This is achieved in Arabidopsis using TILLING to obtain an allelic series of mutants and T-DNA insertion to generate null mutations. The latter uses the efficient transformation system available for this plant, but lacking in our target species, so we need to find alternatives. One such system, named de-TILLING, is based on deletions induced by fast-neutron (FN) mutagenesis. The use of both TILLING and de-TILLING mutation detection systems in a species permits the isolation of an allelic series of mutants including nulls. A third method for studying allelic differences in genes is by using ecotype variation (Eco-TILLING). We have assembled or are in the process of assembling populations for TILLING in L. japonicus, M. truncatula and B. rapa, and a population of FN plants for M. truncatula. Mutation detection is usually carried out using gel-based systems that are inefficient and costly. We have developed an ABI3730 system that is highly efficient. We now wish to gather our existing populations, generate complementary populations and migrate all three mutation detections systems onto the ABI platform for our target species to develop a reverse genetics service for the community. To do this we propose to establish FN populations for L. japonicus and B. rapa, Eco-TILLING populations for all our target species and collate the data in an open-source database available over the web. Under the umbrella of the single technology platform, we will create a unique resource for mutation discovery and functional genomics for the benefit of research community.